The goal of the principal investigator is to continue to develop intellectual, technical, and analytical skills to become an independently funded physician-scientist investigator in examining the alterations in the enteric nervous system in diseases with altered gastrointestinal motility such as Diabetes. The program to achieve this goal will consist of additional didactic and laboratory training in neurobiology, signal transduction and the use of transgenic animals. Diabetes mellitus is a major health concern with a current prevalence of 18.2 million people in the United States. Gastrointestinal dysfunction occurs in as many as 75% of diabetic patients. Injury to the enteric neurons in the setting of diabetes includes neuronal cell death as well as injury to axonal processes. Several studies including preliminary data outlined in this proposal have shown that the phosphotidyl 3-kinase (PI-3 kinase) pathway plays a central role in promoting neuronal growth. However, the mechanism(s) by which diabetes leads to neuronal injury is poorly understood. The overall aim of my proposal is to characterize the role of PI-3 kinase/AKT signaling pathway in the survival of neurons under normoglycemic and hyperglycemic conditions in culture and in vivo in animal models. The following three specific aims are designed to test the hypothesis that hyperglycemia decreases PI-3 kinase mediated signaling pathway activity, which leads to enteric neuronal apoptosis and enteric neuropathy. Specific aim 1: To evaluate the role of the PI-3 kinase pathway in growth factor mediated enteric neuronal survival, proliferation and process extension. The importance of this pathway related to cell survival, proliferation and neuronal process extension will be demonstrated using dominant negative constructs and small interfering RNA. Specifie aim 2: To examine the role of the PI-3 kinase pathway on the hvperglycemia-induced effects on neuronal survival. Preliminary data shows that enteric neuronal precursors cultured in the presence of high glucose concentrations (25mM and 40mM) undergo significant apoptosis and a decrease in the PI-3 kinase activity as evidenced by decreased Akt phosphorylation and increased forkhead translocation. Studies will be carried out to examine the role of various concentrations of glucose (5mM to 40mM) on enteric neuronal survival, proliferation and process extension and the role of the PI-3 kinase pathway in mediating these effects. Specific aim 3: To examine the PI-3 kinase pathwav in the enteric neurons of a diabetic mouse model. This aim represents an in-vivo correlation of experiments proposed in Specific Aim 2. Studies will be performed using the streptozotocin-induced mouse model of diabetes. The effect of diabetes on enteric neuronal number, gastrointestinal motility, and PI-3-Kinase signaling will be assessed. The role of GDNF over expression on the changes induced by diabetes will be determined using two models: a transgenic animal model over expressing GDNF in the glial cell and by daily subcutaneous administration of GDNF. Clarifying the normal role of these signal transduction pathways within developing enteric neurons and determining how alterations in glucose levels influence these signal transduction pathways may provide novel therapeutic strategies to prevent the neuronal injury that occurs in patients with chronic diabetes.